Shear-thinning hydrogel, kit and method of preparation

ABSTRACT

A shear-thinning hydrogel composition includes: a first polymer chain including: (i) a first plurality of units each having at least one of a monosaccharide and an amino acid; and (ii) a cross-linking group bound to the at least one of the monosaccharide and the amino acid of one of the first plurality of units via conversion of a carboxyl group of the unit to a peptide bond; a second polymer chain including a second plurality of the units; and a cross-linking additive connecting one of the second plurality of units to the first polymer chain via the cross-linking group.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.16/509,739, filed Jul. 12, 2019, which is a divisional application ofU.S. Ser. No. 15/637,002, filed Jun. 29, 2017, which is a continuationof PCT application number PCT/IB2017/050460, filed Jan. 27, 2017, whichclaims priority to U.S. 62/288,778, filed Jan. 29, 2016, all of whichare incorporated herein by reference.

FIELD

The specification relates generally to hydrogels, and specifically to ashear-thinning hydrogel, as well as a kit and method for preparing theshear-thinning hydrogel.

BACKGROUND

Hydrogels have many applications in the fields of medicine and lifesciences, particularly in tissue engineering and regenerative medicine(TERM). Tissue engineering involves the construction or reconstructionof animal (e.g. human) tissue using cells and other components foundwithin tissue, such as extracellular matrix components. A goal of TERMis to develop transplantable tissue and organs to address issuesassociated with organ transplants, such as organ shortage and organrejection. TERM can enable medical professionals to use a patient's owncells to create new tissue and organs for the patient.

Regenerative medicine is the clinical application of this TERMtechnology for the purpose of regenerating damaged organs or tissue andfor conducting implants and transplants using engineered tissue ororgans. A goal of regenerative medicine is to use a patient's own cellsto create transplantable tissue and organs to address the issues oforgan shortage and immune system rejection that occur when transplantingdonated organs.

The field of tissue engineering also includes tumor engineering, the useof cancer cells to create tumors for testing and research purposes, aswell as personalized medicine. These tumors are three-dimensionalaggregates of cells which attempt to mimic the conditions that occur incancer in humans and other animals. Such tumors can be used to testcancer drugs in vitro, and may allow for more accurate testing by bettersimulating the conditions under which tumours develop within animals(e.g. by simulating cell-cell and cell-matrix interactions and byproviding an extracellular matrix).

Hydrogels are employed in tissue engineering and regenerative medicine,for example for the formation of three-dimensional biological scaffoldsin vitro and in vivo, and in some cases ex vivo. Some applicationsrequire that the hydrogels be injectable, such as the use of 3Dbioprinters and other injection devices to build the scaffolds,transplant organs, and the like. Further, such hydrogels typically arealso expected to provide a certain degree of mechanical strength andstiffness to support cell growth.

The conflicting requirements of injectability (preferably withoutdamaging cells or other materials suspended within the hydrogel) andmechanical strength can render the design of such hydrogels difficult.Physical cross-linking, for example, may be employed to provide somemechanical strength while still allowing the hydrogel to be injected.For example, PCT patent publication no. WO 2014028209 A1 describes ahydrogel with a guest-host cross-linking mechanism, which softens orliquefies under pressure (i.e. undergoes shear-thinning) to allowinjection. PCT patent publication no. WO 2011084710 A1 discusses anothertype of cross-linking, based on metal-ligand complexes.

However, physical cross-linking may not have sufficient mechanicalstrength to provide a suitable extracellular matrix for the generationof simulated tumours and other structures.

SUMMARY

According to an aspect of the specification, a shear-thinning hydrogelcomposition is provided, comprising: a first polymer chain including:(i) a first plurality of units each having at least one of amonosaccharide and an amino acid; and (ii) a cross-linking group boundto the at least one of the monosaccharide and the amino acid of one ofthe first plurality of units via conversion of a carboxyl group of theunit to a peptide bond; a second polymer chain including a secondplurality of the units; and a cross-linking additive connecting one ofthe second plurality of units to the first polymer chain via thecross-linking group.

According to another aspect of the specification, a kit is provided,comprising: a quantity of a powdered polymer including: (i) a pluralityof units each having at least one of a monosaccharide and an amino acid;and (ii) a cross-linking group bound to the at least one of themonosaccharide and the amino acid of one of the first plurality of unitsvia conversion of a carboxyl group of the unit to a peptide bond; and aquantity of a cross-linking additive for connecting first and secondchains of the polymer via the cross-linking group.

According to a further aspect of the specification, a method ofpreparing a shear-thinning hydrogel composition is provided, comprising:preparing a solution of a first polymer chain including: (i) a firstplurality of units each having at least one of a monosaccharide and anamino acid; and (ii) a cross-linking group bound to the at least one ofthe monosaccharide and the amino acid of one of the first plurality ofunits via conversion of a carboxyl group of the unit to a peptide bond;and mixing, into the aqueous solution, a cross-linking additive tocross-link first and second chains of the polymer.

BRIEF DESCRIPTIONS OF THE DRAWINGS

Embodiments are described with reference to the following figures, inwhich:

FIG. 1 depicts a shear-thinning hydrogel, according to a non-limitingembodiment;

FIG. 2 depicts a unit of a polymer chain, according to a non-limitingembodiment;

FIG. 3 depicts a unit of a polymer chain, according to anothernon-limiting embodiment;

FIG. 4 depicts a cross-linking group bound to the unit of FIG. 3,according to a non-limiting embodiment;

FIG. 5 depicts a shear-thinning hydrogel, according to anothernon-limiting embodiment;

FIG. 6 depicts a shear-thinning hydrogel, according to a furthernon-limiting embodiment;

FIG. 7 depicts a cross-linking group bound to the unit of FIG. 3,according to another non-limiting embodiment;

FIGS. 8-11 depict the formation of a cross-link with the hydrogel unitof FIG. 7, according to a non-limiting embodiment;

FIGS. 12-15 depict NMR plots for various components of a shear-thinninghydrogel, according to a non-limiting embodiment;

FIG. 16 depicts a method of preparing a shear-thinning hydrogel,according to a non-limiting embodiment.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present disclosure is directed to shear-thinning hydrogels. Ingeneral, the hydrogels described herein include a plurality of polymerchains, including hydrophilic polymer chains, including at least a firsthydrophilic polymer chain and a second hydrophilic polymer chain. Eachchain includes a plurality of repeating units; as will be discussedbelow, in some embodiments, each chain includes a plurality of unitseach containing at least one of a monosaccharide and an amino acid. Oneor more of the units of either or both of the first chain and the secondchain includes a cross-linking group. In some embodiments, in which theunits of the chain each include a monosaccharide, the cross-linkinggroup can be bound to the monosaccharide or the amino acid viareplacement of a carboxyl group by a peptide bond.

The hydrogel also includes a cross-linking additive, various examples ofwhich will be discussed herein. The cross-linking additive connects aunit of the second chain mentioned above to a unit of the first chain.More specifically, the cross-linking additive interacts with across-linking group on each chain to establish a cross-link between thechains, thus imparting greater stiffness to the hydrogel than wouldexist in the absence of the cross-link. As will also be discussed below,the resulting hydrogel is also shear-thinning in at least certain stagesof the preparation thereof.

Referring now to FIG. 1, a shear-thinning hydrogel 100 is depictedaccording to certain embodiments. The hydrogel 100 includes first andsecond hydrophilic polymer chains 104-1 and 104-2 (which may also befirst and second segments of the same polymer chain), each including atleast one cross-linking group 108-1, 108-2. The nature of the polymerchains 104 and the cross-linking groups 108 will be described below ingreater detail. In addition, the hydrogel 100 includes a cross-linkingadditive 112 that establishes bonds 116 between respective pairs ofcross-linking groups. As a result, each molecule of the cross-linkingadditive 112 establishes at least one cross-link between the first andsecond chains 104-1 and 104-2.

When sufficient pressure is applied to the hydrogel 100, as shown on theright side of FIG. 1, at least a subset of the bonds 116 are broken, andthe hydrogel 100 transitions to an injectable state with a lowerstiffness than the gel state shown on the left side of FIG. 1. Uponremoval of the above-mentioned pressure (e.g. after injection iscomplete), the cross-linking additive molecules 112 are free tore-establish the bonds 116 with the cross-linking groups 108, and thehydrogel 100 returns to the stiffer (relative to the injectable state)gel state shown on the left side of FIG. 1. As will be discussed ingreater detail herein, in other embodiments the bonds 116 established bythe cross-linking additive 112 are unaffected by the application ofpressure. Instead, in such embodiments, other cross-linking bonds (notshown in FIG. 1) are broken by the application of pressure.

As noted above, in some embodiments the hydrophilic polymer includes aplurality of repeating units each including a monosaccharide. Turning toFIG. 2, in an embodiment, the first and second polymer chains 104-1 and104-2 include alginate (i.e. alginic acid) chains, and thus include aplurality of repeating units 200 each including a monosaccharide. In theembodiment shown in FIG. 2, each repeating unit 200 includes twosaccharides, 204-1 and 204-2. Further, in the illustrated embodiment themonosaccharide (both 204-1 and 204-2 in this example) includes acarboxyl group 208.

Turning to FIG. 3, in other embodiments the first and second polymerchains 304-1 and 304-2 include hyaluronic acid. Thus each polymer chain304 includes a plurality of repeating units 300. As with the embodimentof FIG. 2, each of the units 300 includes a monosaccharide; morespecifically, the repeating unit 300 is a disaccharide (i.e. includingtwo monosaccharides 304-1 and 304-2). Further, in the illustratedembodiment the monosaccharide (304-1 in this example) includes acarboxyl group 308.

In other embodiments (not shown), the polymer chains comprising thehydrogel 100 include a combination of hyaluronic acid and alginate. Forexample, in some embodiments the hydrogel 100 includes equal parts (bymass) hyaluronic acid and alginate. In other embodiments, the hydrogel100 includes one part (by mass) of hyaluronic acid for two partsalginate. In further embodiments, the hydrogel can be composed entirelyof hyaluronic acid, entirely of alginate, or of any intermediate mixtureof the polymers. In embodiments in which alginate and hyaluronic acidare combined, either one of, or both of, the alginate and hyaluronicacid can include the cross-linking features discussed below.

In further embodiments, other polymers may be employed, including anyone of, or any suitable combination of, agarose, alginate, RGD-modifiedalginate, chitosan, collagen, dextran, fibrin, gelatin, GelMA, glycogen,heparin, polyethylene glycol, poly(glycolic acid), poly(lactic acid),and poly(lactic acid-glycolic acid).

As also noted earlier, one or more of the units 200, 300 of each of thepolymer chains 104-1, 104-2 include a cross-linking group 108. Turningnow to FIG. 4, in some embodiments in which hyaluronic acid is employedfor at least a subset of the polymer chains in the hydrogel 100, thecross-linking groups 108 are each connected to units of the hyaluronicacid by replacement of the carboxyl group 308 (R—COOH) with a peptidebond (R—CONH). More specifically, as shown in FIG. 4, a modified versionof the unit 300, labelled 300′, is depicted with a cross-linking groupconnected by replacement of the carboxyl group with a peptide bond 400.In the example embodiment illustrated in FIG. 4, the cross-linking group108 includes a ligand, such as a catechol group 404.

A variety of molecules can be employed to modify the unit 300 (or,indeed, the unit 200 or other suitable polymer units as will occur tothose skilled in the art) to provide the catechol group 404. In theexample of FIG. 4, the unit 300 has been modified with a molecule ofdopamine to provide the catechol group 404. Other molecules can also beemployed to provide the catechol group 404. For example, in someembodiments, the catechol group 404 is applied by bonding of a moleculeof dopa to the unit 300. In still further embodiments, the catecholgroup 404 is applied by bonding of a molecule of epinephrine to the unit300. Various other modifying molecules bearing catechol groups (e.g.norepinephrine) will also occur to those skilled in the art. In someembodiments, combinations of the above-mentioned molecules are employedto apply catechol groups to the polymer chains.

Further, various techniques will now be apparent to those skilled in theart for carrying out the modification of the base polymer with thecatechol group 404, prior to preparation of the hydrogel 100.

In embodiments employing the catechol group 404 as the cross-linkinggroup, the cross-linking additive 112 is a substance that binds to thehydroxyl groups of the catechol group 404. In the present embodiment,the cross-linking additive comprises a metal ion to be mixed with themodified base polymer (e.g. hyaluronic acid modified with dopamine, asset out above). Upon addition of the metal ion, typically in solution,to the polymer (which is typically dissolved in water,phosphate-buffered saline or the like), each metal ion establishes bondswith one or more cross-linking groups 108. Where two or morecross-linking groups are bonded to a given metal ion, cross-links areestablished between the corresponding polymer chains carrying thecross-linking groups.

Various metal ions are contemplated for use as the cross-linkingadditive 112. For example, the metal ion can be selected from iron,aluminum, chromium, copper and manganese. In some embodiments, acombination of metal ions can be employed as the cross-linking additive112. In some embodiments, the oxidative state of the metal ion is 3+. Inother embodiments, the oxidative state of the metal ion is 2+. Infurther embodiments, the oxidative state of the metal ion is 4+ (e.g.for manganese, titanium or the like). For metal ions with a 3+ state,each atom in the metal ion solution is able to form one, two or threelinks with respective catechol groups 404. Turning to FIG. 5, an examplehydrogel 100 is shown in which a third polymer chain 104-3 (e.g. ahyaluronic acid chain having catechol groups bonded thereto ascross-linking groups 108) is cross-linked to both the chains 104-1 and104-2 by a cross-linking additive particle (e.g. Fe³⁺).

The number of cross-linking groups 108 to which each metal ion bonds ispH-dependent, due to the effect of the pH of the hydrogel 100 on thehydroxyl groups of the catechol group 404. More specifically, theApplicant has found that when the hydrogel 100 has a pH belowapproximately 5, each iron ion is most likely to bond with zero or onecatechol groups (i.e. more likely than to bond with two or threecatechol groups). When the hydrogel has a pH between approximately 5 andapproximately 9, each iron ion is most likely to bond with two catecholgroups, as shown in FIG. 1 and in one of the bonds of FIG. 5. Further,when the hydrogel has a pH above approximately 9, each iron ion is mostlikely to bond with three catechol groups, as shown in the left portionof FIG. 5.

Therefore, the pH of the hydrogel 100 is adjusted to obtain the desiredlevel of metal-ligand cross-linking. In the present embodiment, the pHof the hydrogel 100 is adjusted (e.g. by addition of an acid such ashydrochloric acid, or a base such as sodium hydroxide) to a pH ofbetween approximately 5 and approximately 9. For certain applications, ahydrogel with a greater stiffness may be desired, in which case the pHof the hydrogel 100 may be adjusted to above 9.

In further embodiments, other substances are contemplated for use as thecross-linking additive 112, and therefore cross-linking groups 108 otherthan the above-mentioned catechol groups 404 are employed. As notedearlier, in some embodiments the bonds 116 formed by the cross-linkingadditive 112 may not be those that break under pressure; instead, othertypes of cross-links may provide the shear-thinning property of thehydrogel.

Accordingly, turning to FIG. 6, a shear-thinning hydrogel 600 is shownaccording to another embodiment. The hydrogel 600 includes a firsthydrophilic polymer chain 604-1 and a second hydrophilic polymer chain604-2, which are substantially as described above in connection withchains 104-1 and 104-2. In the present embodiment, it is contemplatedthat chains 604-1 and 604-2 are hyaluronic acid chains, withmodifications as set out below. In other embodiments, various otherpolymers can be employed for chains 604-1 and 604-2, including alginateand mixtures of hyaluronic acid and alginate.

The chains 604 include respective cross-linking groups 608-1 and 608-2,as well as a cross-linking additive 612 configured to cross-link thechains 604 via bonds 616 with the cross-linking groups 608. As set outearlier, the cross-linking groups 608 are connected to the polymerchains 604 via the replacement of a carboxyl group with a peptide bond.Referring to FIG. 7, a unit 700 of hyaluronic acid is depicted with anamine cross-linking group 704 connected thereto by a peptide bond 708and a carbon chain 712. The length of the chain 712 need not be exactlyas shown.

Returning to FIG. 6, as illustrated in the lower-right portion of thedrawing, the cross-linking additive 612 establishes bonds 616 withcross-linking groups 608 on each of the first and second chains 604 tocross-link the chains 604. In the present embodiment, in which thecross-linking groups 608 are amine groups as illustrated in FIG. 7, thecross-linking additive 612 is a bridging molecule such as genipin, eachmolecule of which can interact with two amine groups to form across-link between the chains 604. FIGS. 8-9 depict the bonding of amolecule of genipin to a first amine group (e.g. on the chain 604-1),and FIGS. 10-11 depict the bonding of that molecule of genipin to asecond amine group (e.g. on the chain 604-2). As will be apparent tothose skilled in the art, the illustrated units of the first and secondpolymer chains conduct nucleophilic attacks on the molecule of genipin,resulting in a crosslink between the polymer chain units.

In other embodiments, the cross-linking additive includes othermolecules; for example rose bengal is employed as a cross-linkingadditive in certain embodiments. In those embodiments, the cross-linkinggroups include furan groups, connected to the polymer chain via apeptide bond, rather than the amine groups discussed above. Further, insuch embodiments rose bengal acts as a photo-initiator, bonding polymerchains via the above-mentioned furan groups in the presence of visiblelight, without becoming part of the bonds itself.

Referring again to FIG. 6, in the embodiment discussed above, in whichthe cross-linking additive 612 is genipin, the bonds 616 typically donot break under the application of pressure (e.g. during injection ofthe hydrogel 600). In this embodiment, the shear-thinning property isinstead provided at an earlier stage of the preparation of the hydrogel600. More specifically, the first chain 604-1 includes at least oneguest cross-linking group 624. In the present example, the guestcross-linking group 624 is connected to a unit of the second chain viaesterification. Further, the second polymer chain 604-2 includes atleast one host cross-linking group 620 connected to the chain 604-2, forexample, by the replacement of a carboxyl group with a peptide bond.Thus, some units of the chain 604-2 include a host cross-linking group,while other units include an amine group as discussed above.

More specifically, in some embodiments the host cross-linking group 620is cyclodextrin, and the guest cross-linking group 624 is adamantane.Other suitable host and guest groups will also occur to those skilled inthe art. As seen in FIG. 6, cyclodextrin and adamantane bond to eachother to form a guest-host complex 628, which may be broken underpressure, thus providing a shear-thinning property to the hydrogel 600.When genipin is employed as the cross-linking additive, the addition ofgenipin begins a substantially irreversible stiffening process in thehydrogel 600, with the secondary cross-linking bonds 616 providingadditional mechanical strength to the hydrogel 600.

Various other embodiments of the hydrogels discussed above are alsocontemplated. For example, in other embodiments, the amine-genipincross-links of the hydrogel 600 can be replaced by another cross-linkingmechanism, such as the metal-ligand mechanism discussed earlier herein.Further, in some embodiments both of the types of cross-linking additivediscussed above can be employed, with or without the use of a guest-hostmechanism. In other words, in addition to (optionally) the guest andhost complexes, each polymer chain can include respective subsets ofunits carrying amine groups and catechol groups.

The hydrogels 100 and 600 are prepared, in certain embodiments, from akit of materials. The kit includes a quantity of a powdered hydrophilicpolymer including a plurality of units each having a monosaccharide, anda cross-linking group bound to the monosaccharide of one of the firstplurality of units via conversion of a carboxyl group of the unit to apeptide bond. That is, the kit includes at least an amount of powderedpolymer such as alginate or hyaluronic acid (or a combination of the twopolymers), modified with a cross-linking group. In embodiments employinga guest-host mechanism as shown in FIG. 6, the kit includes twoseparately-stored quantities of powdered modified hydrophilic polymer.

The first quantity includes the polymer (modified with the cross-linkinggroup as mentioned above) further modified with a host cross-linkinggroup such as cyclodextrin. The second quantity includes the polymer(modified with the cross-linking group as mentioned above) furthermodified with a guest cross-linking group compatible with the hostgroup, such as adamantane.

As noted earlier, techniques for the preparation of the variousmodifications to base polymer chains are familiar to those skilled inthe art. The modifications may be verified, for example by way ofnuclear magnetic resonance (NMR) studies. FIGS. 12, 13, 14 and 15 depictNMR results for chains of hyaluronic acid modified with, respectively,cyclodextrin, adamantane, dopamine (employing dimethyl sulfoxide, DMSO,for synthesis of the modified polymer), and dopamine (employing aqueoussynthesis of the modified polymer).

The kit further includes a quantity of the cross-linking additive (e.g.a metal ion in solution, genipin or other suitable additive). Whenmultiple additives are to be employed, the kit may includeseparately-stored quantities of each cross-linking additive.

In some embodiments, the kit also includes an amount of a fillerpolymer, which is not modified as discussed above. For example, the kitcan include a quantity of powdered modified hyaluronic acid, and afurther quantity of unmodified powdered alginate.

Turning to FIG. 16, a method 1600 of preparing the hydrogel 100, 600 isdepicted according to certain embodiments. At block 1605, a quantity ofpolymer modified with a cross-linking group is dissolved in water,tris-buffered saline (TBS), phosphate-buffered saline (PBS), or anyother suitable solvent. When a guest-host mechanism is employed, atblock 1605 the two quantities may be dissolved together or separatelyand then mixed together. In embodiments in which a filler polymer isemployed, the filler polymer is also dissolved at block 1605, eitherseparately from the modified polymer for subsequent mixing, or togetherwith the modified polymer.

In some embodiments, the amount of polymer is less than about 15 percentby mass of the hydrogel. In other embodiments, the amount of polymer isless than about 10 percent by mass of the hydrogel. In furtherembodiments, the amount of polymer is less than about 5 percent by massof the hydrogel. Further, in embodiments in which a guest-host mechanismis employed, the guest and host-modified polymers are typically employedin equal proportions. In other embodiments, the guest and host-modifiedpolymers are employed in different proportions. Where a filler (i.e.unmodified) polymer is employed, the filler polymer can represent up totwice the total amount of polymer in the hydrogel than the modifiedpolymer. Thus, an example hydrogel prepared according to the method 1600may include 5% by mass modified hyaluronic acid, and 10% by massunmodified alginate. Further, the cross-linking additive is typicallyless than 10% by mass of the hydrogel.

The preparation of the dissolved polymer at block 1605 includes heatingthe solution during preparation to accelerate dissolution of the polymerpowder(s), in some embodiments. For example, the solution may be heatedto about 30° C. in some embodiments. In other embodiments, the solutionmay be heated up to, but not beyond, the degradation temperature of thepolymer (e.g. about 60° C. for hyaluronic acid).

Following the performance of block 1605, the mixture is allowed to restfor a period of time. For example, the mixture may be left for 24 hours,although greater or smaller time periods may also be employed. Block1610 can also be omitted in some embodiments, particularly those notemploying a guest-host mechanism.

After the performance of block 1610 (or block 1605, if block 1610 isomitted), the method 1600 proceeds to either of block 1610 a or 1610 b.At block 1610 a, the cross-linking additive is added to the solution,and then at block 1615 a the resulting hydrogel is dispensed via anysuitable mechanism. For example, syringes, pipettes, 3D printers,syringe pumps, dispensing machines, high-throughput screening systems,and automated or manual injection and extrusion systems may be employedat block 1615 a.

As will now be apparent, the addition of cross-linking additive at block1610 a begins the mechanical strengthening of the hydrogel via theformation of cross-linking bonds (in addition to those formed byguest-host complexes at block 1610, if such complexes are employed). Asnoted earlier, when the cross-linking additive is genipin, the resultingcross-links are typically not breakable under pressure, and thereforethe performance of block 1615 a typically follows the performance ofblock 1610 a closely in time (e.g. within about thirty minutes) for suchmaterials.

Further, when the cross-linking additive is a metal ion, the resultingcross-links typically form more quickly than those formed by genipin.Further, the resulting metal-ligand bonds may be more resistant tobreaking under pressure than the guest-host bonds. Therefore, when ametal-ligand mechanism is employed, the performance of block 1615 atypically follows the performance of block 1610 a closely in time (e.g.within about five minutes).

Alternatively to the performance of blocks 1610 a and 1615 a, theperformance of method 1600 can instead proceed from block 1610 (or 1605,when block 1610 is omitted) to block 1610 b. At block 1610 b, cells(e.g. tumour cells) are added to the hydrogel as needed, and thehydrogel is dispensed as described above. The cross-linking additive isthen added to the hydrogel after dispensing. For example, a metal ion insolution may be added to each well in a 96-well plate after the wellshave been filled with hydrogel suspending tumour cells. The performanceof blocks 1610 b and 1615 b may be desirable for fast-actingformulations, such as those employing the metal-ligand mechanism. Infurther embodiments, the performance of method 1600 can include theaddition of both cells and cross-linking additive(s), followed bydispensing the hydrogel.

Various applications may make use of hydrogels as described herein. Forexample, the hydrogels described above may be employed in personalizeddrug testing, such that a plurality of simulated tumours (e.g. in a96-well plate) may be seeded with patient cells supported within ahydrogel matrix. Various different prospective drugs may then be appliedto the wells, and their effects on cell viability monitored (e.g. viaATP assays).

Certain advantages to the hydrogels described herein will be apparent tothose skilled in the art. For example, the use of hyaluronic acid incertain embodiments may better simulate the biological environment ofcells (as hyaluronic acid also appears in the extracellular environmentin vivo).

The scope of the claims should not be limited by the embodiments setforth in the above examples, but should be given the broadestinterpretation consistent with the description as a whole.

The invention claimed is:
 1. A kit comprising: a quantity of a powderedpolymer including collagen and a first polymer chain, the first polymerchain including: (i) a first plurality of units of hyaluronic acid, eachunit having at least one monosaccharide; and (ii) a further plurality ofunits of alginate bound to the first plurality of units; and (iii) across-linking group comprising dopamine bound to the at least onemonosaccharide of one of the first plurality of units via conversion ofa carboxyl group of the unit to a peptide bond; and a quantity of across-linking additive comprising a 2+ charged ion for connecting firstand second chains of the polymer via the cross-linking group, the secondchains of the polymer including a second plurality of the units, thecross-linking additive connecting one of the second plurality of unitsto the first polymer chain via the cross-linking group.
 2. The kit ofclaim 1, wherein the quantity of the powdered polymer includes: a firstquantity of a first powdered polymer, including a host cross-linkinggroup bound to another of the first plurality of units via conversion ofa carboxyl group of the other unit to a peptide bond; and a secondquantity of a second powdered polymer, including a guest cross-linkinggroup bound to one of the second plurality of the units, for bindingwith the host cross-linking group.
 3. The kit of claim 1, wherein thecross-linking group comprises a first ligand group; and wherein the oneof the second plurality of units comprises a second ligand group, thecross-linking additive connecting to each of the first ligand group andthe second ligand group.
 4. The kit of claim 3, wherein the ligand groupand the second ligand group each comprise a catechol group.
 5. The kitof claim 1, wherein the kit further comprises an amount of fillerpolymer.
 6. The kit of claim 1, wherein the kit further comprises anamount of at least one of: water, tris-buffered saline, andphosphate-buffered saline.
 7. The kit of claim 1, wherein the first andsecond polymer chains are to represent less than 15% by mass of ashear-thinning hydrogel composition prepared by the kit.
 8. The kit ofclaim 1, wherein the first and second polymer chains are to representless than 10% by mass of a shear-thinning hydrogel composition preparedby the kit.
 9. The kit of claim 1, wherein the first and second polymerchains are to represent less than 5% by mass of a shear-thinninghydrogel composition prepared by the kit.
 10. The kit of claim 1,wherein the 2+ charged ion comprises Fe²⁺.